This invention relates to Plasma Protein Fraction (Human) substantially free of bradykinin, kininogen, and activators of prekallikrein, and to methods for producing same.
For a number of years, solutions of Plasma Protein Fraction (Human) have enjoyed extensive use in the treatment of shock, hypoproteinemia and other conditions requiring the use of plasma expanders. Plasma Protein Fraction (Human) is the official nomenclature which has been adopted in the U.S. by the FDA (21 CFR 640.90) for the product as obtained by the modified Cohn fractionation process applied to human plasma as disclosed in U.S. Pat. No. 2,958,628. Plasma Protein Fraction (Human) also called PPF (Human), is a mixture of constituent plasma proteins and under 21 CFR 640.90 shall consist of at least 83% albumin and no more than 17% globulins of which no more than 1% shall be gamma globulin. The globulins are a mixture of .alpha.- and .beta.-globulins, and actually only those species of .alpha.- and .beta.-globulins having molecular weights similar to that of albumin (see U.S. Pat. No. 2,958,628). The constituent plasma proteins in Plasma Protein Fraction (Human) therefore make up a substantially unique mixture which differs from other plasma fractionation products obtained by different processes even though these other products may include albumin and .alpha.- and .beta.-globulins.
Vasodepressor effects have sometimes been noted when PPF solutions were infused rapidly or used in cardiopulmonary bypass procedures, a condition which could by extremely hazardous to a patient. The presence of bradykinin in such solutions was implicated according to Izaka et al (Transfusion 14, pp. 242-248, 1974) and methods for removing bradykinin from PPF by physical means were disclosed in U.S. Pat. No. 3,876,775. Such means included exposing the PPF solutions to silica gel or cation exchange resins to adsorb the bradykinin. These methods have the disadvantage of also adsorbing some of the desired plasma proteins and thereby reducing the yield of PPF.
It has been observed in our laboratories that certain lots of PPF solution which were shown to be substantially free of bradykinin, nevertheless caused a depressor effect when infused rapidly into dogs. Investigation of such solutions revealed that they had significant amounts of kininogen present. It was postulated that this precursor was converted into bradykinin by the action of kallikrein or other proteases circulating in the dog's blood stream. Patients in shock, particularly those in which shock is brought about by endotoxins or pancreatitis, could be seriously compromised if Plasma Protein Fraction containing kininogen were rapidly infused since their blood pressure is already greatly depressed.
In U.S. Pat. No. 4,017,470, Izaka et al, there is described a method for the production of a heat-stable plasma protein fraction and it is stated the product has no blood pressure depressing action. The product is disclosed as containing 93-95 percent albumin and 5-7 percent of alpha globulin. The product is derived from Cohn's Fraction IV-1 by a process which includes (1) heating a solution of Fraction IV-1 at pH 4.5 to 5.5 at 50.degree.-65.degree. C. with an organic acid for 1 to 4 hours to precipitate most of the unstable lipo- and glycoproteins; (2) treating the supernatant with Rivanol.RTM. to precipitate the residual lipoprotein, and (3) adsorbing peptide substances showing blood pressure-depressing action (by rat uterus contraction test) which are formed from blood pressure-depressing substances such as kininogen with an inorganic adsorbent (e.g., silica gel or cation exchanger). This third step is, of course, the same as that disclosed in U.S. Pat. No. 3,876,775 (supra), which describes the removal of bradykinin from Plasma Protein Fraction. To avoid any confusion from statements in the U.S. Pat. No. 4,017,470 which refer to "blood pressure-depressing substances such as kininogen", it is well known that kininogen itself exhibits no depressor effect in the rat uterus test; hence, Izaka et al obviously intended such statements to mean kininogen has the potential for producing depressor substances. In any event, there is no indication that their product is free of kininogen. Moreover, their product is not Plasma Protein Fraction but quite a different mixture whose constituent plasma proteins differ significantly from those of PPF.
Recently, it was noted in our laboratories that occasional production lots of PPF resulted in solutions which caused a depressor effect when rapidly infused into patients. These particular lots were demonstrated to be essentially free of bradykinin, and kininogen was at levels which were considered to contribute little if any toward this depressor action. Further investigation of these solutions showed the presence of significant amounts of activator(s) of prekallikrein (PKA). It was postulated that the PKA in these solutions when injected would then activate the endogenous prekallikrein and lead to the formation of bradykinin in the blood stream.
The sequence of events which is generally postulated for the generation of bradykinin is expressed by the following: ##STR1## In the Cohn method of fractionating plasma, or modifications thereof, some Factor XIIa is most likely generated, some of which may become altered or fragmented. It is believed these altered or fragmented species, as well as intact activated Hageman Factor (Factor XIIa), cause the conversion of prekallikrein to kallikrein. Varying amounts of kininogen, depending on slight changes in the process, are converted by the action of kallikrein into the potent hypotensive substance, bradykinin.
It would be an important improvement if solutions of Plasma Protein Fraction could be consistently prepared which were substantially free not only of bradykinin but also of activators of prekallikrein and kininogen. Quite recently the problem was similarly expressed by Culliver et al (Vox. Sang. 36, pp. 201-207, 1979), although no solution to the problem was offered.
Auerswald et al disclose in U.S. Pat. No. 3,100,737 a heat stable plasma fraction consisting of albumin and alpha and beta globulins derived from plasma by a process similar to the process of the U.S. Pat. No. 4,017,470 but quite different from the modified Cohn fractionation process of the U.S. Pat. No. 2,958,628. Following the removal of fibrinogen and gamma globulin by ammonium sulfate precipitation, the supernatant is mixed with fatty acids and heated at 55.degree. C. for 20 minutes at pH 5.2 to precipitate unstable alpha and beta globulins. In one example, the ammonium sulfate is removed by passing the second supernatant over cationic and anionic resin exchangers which conceivably might also remove any bradykinin present in the final product. Apart from the belief that their product would have constituent plasma proteins differing from those in PPF since the relative processes are so different, there is no indication that their product would be substantially free of either kininogen or activators of prekallikrein.